Research note: Zic5 alters digit morphology during avian limb development

Avian limbs have evolved precise digit lengths and shapes that are optimized for flight and locomotion, but the transcriptional regulators that regulate this architecture remain poorly defined. Although anteriorly biased Zic3 expression constrains digit outgrowth, whether other zinc finger of the cerebellum (Zic) factors-particularly Zic5-shape avian distal limb morphology remains unknown. Here, we define the spatiotemporal dynamics and developmental function of Zic5 expression in the chicken limb and evaluate its role in shaping avian limb and digit morphogenesis. Whole-mount in situ hybridization reveals that Zic5 shows a dynamic, stage-dependent shift from broad distal mesenchymal expression to digit-base-restricted domains in the chicken limb, consistent with a role in constraining digit elongation to shape fore-hind limb morphology. Overexpression of Zic5 in the chicken limb buds resulted in shortened digits and impaired limb ossification. These phenotypes were accompanied with altered expression of skeletal marker genes, including Sox9, Runx2, Col9a1, Col9a3, Ptch2 and Hhip, suggesting that elevated Zic5 expression is sufficient to impair skeletal maturation during limb development. Together, our results indicate that elevated Zic5 expression can impair limb skeletal growth and maturation, with effects on distal digit development that result in digit shortening or loss and suggest that Zic5 may contribute to the regulation of digit architecture during avian morphological diversification.